SysMO-LAB

SysMO is a European transnational funding and research initiative on "Systems Biology of Microorganisms".

The goal pursued by SysMO was to record and describe the dynamic molecular processes going on in unicellular microorganisms in a comprehensive way and to present these processes in the form of computerized mathematical models.

Systems biology will raise biomedical and biotechnological research to a new quality level and contribute markedly to progress in understanding. Pooling European research ...

Basically extending SYSMO-LAB 1st phase into second with addition of fourth species, Lb. plantarum. The main focus is amino acid metabolism. primary metabolisms, like glycolysis is also interest.

Here SYSMO-LAB put all there pre-liminary data files or models ordered per person and project

Challenge: Comparative analyses, as demonstrated by comparative genomics and bioinformatics, are extremely powerful for (i) transfer of information from (experimentally) well-studied organisms to the other organisms, and (ii) when coupled to functional and phenotypic information, insight in the relative importance of components to the observed differences and simalities. The central principle of this proposal is that important aspects of the functional differences between organisms derive not ...

Determination of essential amino acids for Streptococcus pyogenes M49

Lactic acid bacteria generally use homolactic fermentation for generation of ATP. Here we studied the role of Arginine and Glutamine metabolism on the general physiology of the lactic acid bacteria Streptococcus pyogenes. A deletion mutant of glnA (glutamine synthetase) has been constructed in the S. pyogenes M49 591 background. The glnA mutant strain shows decreased growth in low glutamine and excess glutamate conditions and no growth at all in low glutamine and low glutamate conditions. An arcA ...

The reconstruction of the metabolic networks is done by sequence comparison with already annotated genomes of L. lactis, L. plantarum, B. subtilis and E. coli

Here you will find all pre-liminary data

Pyruvate kinase (PYK, EC 2.7.1.40) is a key step in glycolysis converting phosphoenolpyruvate into pyruvate. The activity of PYK is activator-dependent, with the allosteric activation mostly being due to fructose-1,6-bisphosphate (FBP).

Pyruvate formate-lyase (PFL) is an important enzyme in the metabolic pathway of lactic acid bacteria (LAB) and is held responsible for the regulation of the shift between homolactic acid to mixed acid fermentation. PFL catalysis the reversible reaction of acetyl-CoA and formate into pyruvate and CoA. A glycyl radical, who is regenerated within the reaction, is involved; therefore, PFL works only under strictly anaerobic conditions. For its activation, the C-terminal domain has to bind to the ...

The two lactic acid bacteria L. lactis and S. pyogenes were studied with respect to the concentration of intracellular metabolites involved in glycolysis in time upon a glucose pulse. Models that describe this behavior are also constructed

Lactic acid bacteria generally use homolactic fermentation for generation of ATP. Here we studied the role of the lactate dehydrogenase enzyme on the general physiology of the three lactic acid bacteria Lactococcus lactis, Enterococcus faecalis and Streptococcus pyogenes. Surprisingly deletion of the ldh genes hardly affected the growth rate in chemically defined medium, however growth rate was affected in rich medium. Furthermore, deletion of ldh affected the ability for utilization of various ...

The Lactate dehydrogenases (LDH) are key metabolic enzymes in lactic acid bacteria (LAB). The LDH ( E.C. 1.1.1.27) catalyzes the reaction of pyruvate and NADH into lactate and NAD+.We have carried out an experimental and computational study of the effects of fructose-1,6-bisphosphate (FBP), phosphate (Pi) and ionic strength (NaCl concentration) on 3 LDHs from 3 LABs studied at pH 6 and pH 7.

S. pyogenes was grown in rich medium, strongly concentrated and glucose-pulsed in a MES buffer. Intracellular metabolite concentration is followed in time.

S. pyogenes M49 (591), E. faecalis V583, and L. lacis NZ9000 and their isogenic ldh deletion mutants were grown glucose free CDM-LAB medium in BIOLOG phenotype microarray plates PM01 and PM02. With this assay the abilitiy of the strains to grow on 190 different carbon sources was determined in 96 well format.

L. lactis was grown in LAB medium or rich THY medium, strongly concentrated and glucose-pulsed in a MES buffer. Intracellular metabolite concentration is followed in time.

Measurements on Km, Vmax and allosteric activation or inhibition of the heterologously expressed (E. coli) and purifiied main L-lactate dehydrogenase

Measurements on Km, Vmax and allosteric activation or inhibition of the main L-lactate dehydrogenase

Global sensitivity analysis of a kinetic model to determine the sensitivities for each parameter, over a wide parameter range. We used the elementary effects method.

Lumped kinetic model of L. lactis glycolysis, formulated with ordinary differential equations. Simulations are in line with experimental data

Enzymes involved in butanediol formation from pyruvate in L. Lactis and E. faecalis were characterized with respect to their Km's for their substrates and their Vmaxes

L. lactis, E. faecalis and S. pyogenes are referred to as LAB because of the fact that in the presence of glucose lactate is produced as the main fermentation product . This metabolic pathway is relatively inefficient, since only 2 ATP are generated from one glucose molecule. All three LAB possess the genetic make up for mixed acid fermentation, a more effective way of fermentation generating 3 ATP per molecule of glucose. All three genomes reveal (at least) two genes encoding a lactate dehydrogenase ...

L. lactis, S. pyogenes and E. faecalis were grown in C-limited chemostat cultures at various pH's and dilution rates. General flux distribution, yields and other physiological factors were studied.

In this experiment we glucose-pulsed an E. faecalis cultures re-suspended in 100 mM MES buffer at pH 6.5. Samples were taken in time to study intra- and extracellular metabolites. These data are used to construct a kinetic model of the catqabolism of E. faecalis

In this experiment we glucose-pulsed an L. lactiss cultures re-suspended in 100 mM MES buffer at pH 6.5. Samples were taken in time to study intra- and extracellular metabolites. These data are used to construct a kinetic model of the catabolism of E. L. lactis

Determination of essential amino acids for Streptococcus pyogenes

Km values of pyruvate kinase of different organisms without/with allosteric effector molecules collected from literature.

Pyruvate formate-lyase (PFL) is an important enzyme in the metabolic pathway of lactic acid bacteria (LAB) and is held responsible for the regulation of the shift between homolactic acid to mixed acid fermentation. PFL catalysis the reversible reaction of acetyl-CoA and formate into pyruvate and CoA. A glycyl radical, who is regenerated within the reaction, is involved; therefore, PFL works only under strictly anaerobic conditions. For its activation, the C-terminal domain has to bind to the ...

S. pyogenes wildetype, an arcA- and a glnA-deletion mutants were grown in CDM-LAB cultures at pH 6.5 and 7.5 and at a growth rate of 0.05

In order to construct an in vivo-like buffer for S. pyogenes, the intracellular concentrations of Fe, K, Mg, Mn Na, P and S elements were determined via ICP-AES (inductively coupled plasma atomic emissionspectroscopy) method at the Institute of Land Use, University of Rostock. The samples for the analysis were obtained from a steady state culture grown on CDM-LAB with glucose.

Metabolic network of S. pyogenes including primary metabolism, polysaccharide metabolism, purine and pyrimidine biosoynthesis, teichoic acid biosynthesis, fatty acid and phospholipid bioynthesis, amino acid metabolism, vitamins and cofactors

Metabolic network of Enterococcus faecalis including primary metabolism, polysaccharide metabolism, purine and pyrimidine biosoynthesis, teichoic acid biosynthesis, fatty acid and phospholipid bioynthesis, amino acid metabolism, vitamins and cofactors

Despite high similarity in sequence and catalytic properties, the L-lactate dehydrogenases (LDH) in lactic acid bacteria (LAB) display differences in their regulation which may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the effects of fructose-1,6-bisphosphate (FBP), phosphate (Pi) and ionic strength (NaCl concentration) on 6 LDHs from 4 LABs studied at pH 6 and pH 7. We find: (1) The extent of activation by FBP (Kact) ...

L. lactis cultures were grown at different dilution rates in glucose-limited chemostat conditions and were analyzed with respect to physiological parameters. Amino acid consumption, glucose consumption and production of fermentation products were measured in steady-state conditions,

Output of the 3D-structures modeled by comparative modeling tool for LDH enzymes from four LABs (in the PDB format, tarred). Four LABs include Enterococcus faecalis, Lactococcus lactis, Streptococcus pyogenes and Lactobacillus plantarum. Output of the SEEK Model https://seek.sysmo-db.org/models/118.

The modeling was performed against a x-ray structure of LDH from B. stearothermophilis (template, PDH ID: 1LDN).

Output files of phosphate probe binding on the surface of LDH from lactococcus lactis type 1. File with extension XPLOR can be visualized with a program VMD to identify the most favorable position for the phosphate binding. This relates to the Model "Part 4".

The Table represents the simulation results of how the presence of phosphate ions (Pi) in the solution might affect the activity of four LDH enzymes. This includes the algorithmic analysis of the binding energies values computed by the GRID program (see Part 4, model) for each enzyme in presence and absence of FBP molecule at pH 6 and 7. The analysis was performed by using the algorithm proposed in Part 5, model.

L. lactis was grown in rich THY medium, strongly concentrated and glucose-pulsed in a MES buffer. Intracellular metabolite concentration is followed in time.

Lactobacillus plantarum JDM1 differentially expressed proteins, comparison between low and high growth rates

Lactobacillus plantarum ATCC 14917 differentially expressed proteins, comparison between low and high growth rates

Lactobacillus plantarum strains WCFS1, NC8, JDM1 and ATCC 14917 amino acid concentrations mM supernatant, high (D=0.4) and low (D=0.05) growth rates

Heterologous Expression of LDH from L.actis (MG1363) in E. coli DH5α. Assessment of kinetic parameters of LDH to include in a catabolic model

Lactobacillus plantarum, 4 strains (WCFS1, NC8, ATCC 14917, JDM1) chemostat experiments low and high growth rates, OD and DW measurements

Lactobacillus plantarum strains WCFS1, NC8, ATCC14917, JDM1. HPLC end products mM measurements (CDM subtracted) and flux calculations, high and low growth rates.

amino acid auxotrophies as determined for S. pyogenes, E. faecalis, L. lactis and L. plantarum (ATCC, NC8, JDM1 and WCFS1) subsequent inoculation (3x) in CDM

Vmaxes E. faecalis from chemostat cultures, D0.05, D0.15 en D0.4 triplicates for every growthrate, measured in in-vivo-like assay (Goel, Teusink, AEM, 2012) glycolytic enzymes + LDH + acetate/EtOH branch

raw data OD600 of amino acid auxotrophy exps S. pyogenes

raw data OD600

Lactobacillus plantarum WCFS1 differentially expressed proteins, comparison between low and high growth rates

Lactobacillus plantarum NC8 differentially expressed proteins, comparison between low and high growth rates

Here is a kinetic model (in COPASI format) of L. lactis glycolysis.

SBML description of L. lactis glycolysis. Same as the uploaded Copasi file

Preliminary metabolic network of S. pyogenes including primary metabolism, polysaccharide metabolism, purine and pyrimidine biosoynthesis, teichoic acid biosynthesis, fatty acid and phospholipid bioynthesis, amino acid metabolism, vitamins and cofactors. The model still needs to be validated.

Batch and chemostat model of L lactis. Scope of the model is to provide a mechanistic explanation of the switch between mixed acid and homolactic fermentation.

The principles of Stealthy Engineering (Adamczyk et al.: Biotechnology Journal 2012; 7(7):877-83) are illustrated in this model by emulating a cross engineering intervention between L. lactis and S. cerevisiae.

The case study consists of replacing the native glucose uptake system of L. lactis with that native to the yeast S. cerevisiae. A modified version of Hoefnagel et al.’s model of L. lacrtis’ central metabolism was used as starting point. The total functional replacement of the PTS with the ...

A reconstruction of the cellular metabolism of the opportunistic human pathogen Enterococcus faecalis V583 represented as stoichiometric model and analysed using constraint-based modelling approaches

3D structure prediction of LDH enzymes from four LAB by comparative modeling against x-ray structure of LDH from B. stearothermophilis (template, PDB ID: 1LDN). The computation was performed with a protocol that uses "automodel.very_fast" settings of Modeller program (http://salilab.org/modeller/).

Comparison of electrostatic potentials within the allosteric binding sites of LDH enzymes to estimate the binding affinity of the FBP molecule is performed with the PIPSA program. The program uses the structure of enzymes in the PDB format and computed electrostatic potentials in the GRD format.

Computation is performed for the modeled 3D structures of LDH enzymes (in PDB format) with the UHBD program, for pH 6 and pH 7.

Binding energies of phosphate ions to the allosteric and catalytic sites were estimated with a program GRID (http://www.moldiscovery.com/soft_grid.php). The calculations were performed for the modeled LDH structures from four LABs, at pH 6 and 7, in presence and absence of the FBP molecule. The phosphate ion was presented as a probe.

In order to estimate whether Pi has an activatory or an inhibitory effect on the enzymes, the computed probe binding energies (from GRID results, Part 4) were compared with those for the LDH from L. plantarum whose activity is known to be unaffected by Pi.

The binding energies of the Pi probe in the allosteric binding site (AS) and the COO probe in the catalytic binding site (CS) of LDH from L. plantarum were defined as E¬AS,threshold and ECS,threshold, respectively. For the other LDH enzymes, ...

Structural models of the LAB PYKs of L. lactis, L. plantarum, S. pyogenes and E. faecalis including the "best" docking solutions of potential allosteric ligands. The structures were derived by homology modeling based on the template of E. coli and B. stearothermophilus. PYK models and ligands are provided as .pdb files and can be displayed by using the program PyMOL, for instance.

The kinetic model includes sugar uptake, degradation of glucose into pyruvate and the fermentation of pyruvate.

The kinetic model includes sugar uptake, degradation of glucose into pyruvate and the fermentation of pyruvate.

Method extraction of intracellular metabolites in Lactococcus lactis

Method extraction of intracellular metabolites in Lactococcus lactis

Method for transformation of plasmids into Lactococcus lactis

Method for synthesis of LAB-medium sued for the SYSMO-LAB project

Method for analysis of various organic acids in the medium

Protocol for applying a glucose perturbation in Streptococcus pyogenes.

Perturbation of starved cells with glucose. Concentrations of intra- and extracellular metabolites are followed in time.

This HPLC method uses a isocratic method and a RI detector to identify and quantify almost all excreted catabolic metabolites.

A protocol for acidic quenching of lactic acid bacteria used for analyses of intracellular metabolites.

This protocol for applying glucose perturbations works for Lactococcus lactis and Enterococcus faecalis

Presentation at the SysMO-LAB2 meeting in Copenhagen November 2012. Group from Nofima/Norwegian University of Life Sciences, Ås, Norway. Work on four strains of Lactobacillus plantarum, omics-analyses, diversity.

Koen van Grinsven

SysMO-LAB2 meeting in Copenhagen Nov 21-22, 2012

HITS/MCM Presentation at the SysMO-LAB2 meeting in Copenhagen on November, 22.11.2012

Poster presented at the 12th International Conference on Systems Biology (ICSB), Heidelberg/Mannheim, August 28 - September 1, 2011.

SysMO-LAB2 Meeting Copenhagen 2012